The bacterial type III secretion system (T3SS) is a complex multi-protein apparatus that facilitates the secretion and translocation of effector proteins from the bacterial cytoplasm directly into the mammalian cytosol. This complex protein delivery device is shared by over 15 species of Gram-negative human pathogens, including Salmonella spp., Shigella flexneri, Pseudomonas aeruginosa, Yersinia spp., enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp. (Hueck, 1998, Type III protein secretion systems in bacterial pathogens of animals and plants, Microbial. Biol. Rev., 62:379-433; Keyser, et al., 2008, Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria, J. Intern. Med., 264:17-29.) In the opportunistic pathogen P. aeruginosa, the T3SS is the major virulence factor contributing to the establishment and dissemination of acute infections (Hauser, 2009, The type III secretion system of Pseudomonas aeruginosa: infection by injection, Nat. Rev. Microbial., 7:654-65). Four T3SS effectors have been identified in P. aeruginosa strains—ExoS, ExoT, ExoY, and ExoU. ExoS and ExoT are bifunctional proteins consisting of an N-terminal small G-protein activating protein (GAP) domain and a C-terminal ADP ribosylation domain; ExoY is an adenylate cyclase; and ExoU is a phospholipase (see review in Engel and Balachandran, 2009, Role of Pseudomonas aeruginosa type III effectors in disease, Curr. Opin. Microbiol., 12:61-6).
In studies with strains producing each effector separately, ExoU and ExoS contributed significantly to persistence, dissemination, and mortality while ExoT produced minor effects on virulence in a mouse lung infection model, and ExoY did not appear to play a major role in the pathogenesis of P. aeruginosa (Shaver and Hauser, 2004, Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung, Infect. Immum., 72:6969-77). While not a prototypical effector toxin, flagellin (FliC) may also be injected into the cytoplasm of host cells from P. aeruginosa via the T3SS machinery, where it triggers activation of the innate immune system through the nod-like receptor NLRC4 inflammasome. (Franchi, et al., 2009, The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis, Nat. Immunol., 10:241-7; Miao, et al., 2008, Pseudomonas aeruginosa activates caspase 1 through 1paf, Proc. Natl. Acad. Sci. USA, 105:2562-7.)
The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients with lower respiratory and systemic infections caused by P. aeruginosa (Roy-Burman, et al., 2001, Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections, J. Infect. Dis., 183:1767-74). In addition, T3SS reduces survival in P. aeruginosa animal infection models (Schulert, et al., 2003, Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia, J. Infect. Dis., 188:1695-706), and is required for the systemic dissemination of P. aeruginosa in a murine acute pneumonia infection model (Vance, et al., 2005, Role of the type III secreted exoenzymes S, T, and Y in systemic spread of Pseudomonas aeruginosa PAO1 in vivo, Infect. Immun., 73:1706-13). T3SS appears to contribute to the development of severe pneumonia by inhibiting the ability of the host to contain and clear bacterial infection of the lung. Secretion of T3SS toxins, particularly ExoU, blocks phagocyte-mediated clearance at the site of infection and facilitates establishment of an infection (Diaz, et al., 2008, Pseudomonas aeruginosa induces localized immunosuppression during pneumonia, Infect, Immun., 76:4414-21). The result is a local disruption of an essential component of the innate immune response, which creates an environment of immunosuppression in the lung. This not only allows P. aeruginosa to persist in the lung, but it also facilitates superinfection with other species of bacteria.
While several antibacterial agents are effective against P. aeruginosa, the high rates of mortality and relapse associated with serious P. aeruginosa infections, even in patients with hospital-acquired pneumonia (HAP) receiving antibiotics active against the causative strain, reflect the increasing incidence of drug-resistant strains and highlights the need for new therapeutic agents. (See, e.g., El Solh, et al., 2007, Clinical and hemostatic responses to treatment in ventilator-associated pneumonia: role of bacterial pathogens, Crit. Care Med., 35:490-6; Rello, et al., 1998, Recurrent Pseudomonas aeruginosa pneumonia in ventilated patients: relapse or reinfection?, Am. J. Respir. Crit. Care Med., 157:912-6; and Silver, et al., 1992, Recurrent Pseudomonas aeruginosa pneumonia in an intensive care unit., Chest, 101:194-8.) Conventional bacteriostatic and bactericidal antibiotics appear insufficient to adequately combat these infections, and new treatment approaches such as inhibitors of P. aeruginosa virulence determinants may prove useful as adjunctive therapies. Veesenmeyer, et al., 2009, Pseudomonas aeruginosa virulence and therapy: evolving translational strategies, Crit. Care Med., 37:1777-86.
The potential for the type III secretion system as a therapeutic target has prompted several groups to screen for inhibitors of T3SS in various bacterial species, including Salmonella typhimurium, Yersinia pestis, Y. pseudotuberculosis, and E. coli. (Reviewed in Keyser, et al., 2008, Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria, J. Intern. Med., 264:17-29; and Clatworthy, et al., 2007, Targeting virulence: a new paradigm for antimicrobial therapy, Nat. Chem. Biol., 3:541-8). High levels of sequence conservation among various proteins comprising the T3SS apparatus suggest that inhibitors of T3SS in one species may also be active in related species. Broad spectrum activity of T3SS inhibitors identified in a screen against Yersinia has been demonstrated in Salmonella, Shigella, and Chlamydia. Hudson, et al., 2007. Inhibition of type III secretion in Salmonella enterica serovar Typhimurium by small-molecule inhibitors, Antimicrob. Agents Chemother., 51:2631-5; Veenendaal, et al., 2009, Small molecule type III secretion system inhibitors block assembly of the Shigella type III secreton, J. Bacteriol., 191:563-70; Wolf, et al., 2006, Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle, Mol. Microbiol., 61:1543-55.
Screening for P. aeruginosa T3SS inhibitors has been reported, leading to several selective inhibitors of P. aeruginosa. T3SS-mediated secretion, one of which reproducibly inhibits both T3SS-mediated secretion and translocation. Aiello, et al., 2010, Discovery and Characterization of Inhibitors of Pseudomonas aeruginosa Type III Secretion, Antimicrob. Agents Chemother., 54(5): 1988-1999.
Clearly, needs remain for new, potent inhibitors of bacterial T3SS of P. aeruginosa and other bacterial species.